infection, genetics and evolution · plasmodium falciparum anopheles coluzzii anti-anapn1...

Infection, Genetics and Evolution 45 (2016) 138–144

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Infection, Genetics and Evolution

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Research paper

Impact of exposure to mosquito transmission-blocking antibodies onPlasmodium falciparum population genetic structure

Maurice M. Sandeu a,b, Luc Abate a, Majoline T. Tchioffo a, Albert N. Bayibéki b, Parfait H. Awono-Ambéné b,Sandrine E. Nsango b,c, Cédric B. Chesnais d, Rhoel R. Dinglasan e,f, Thierry de Meeûs g, Isabelle Morlais a,b,⁎a UMR MIVEGEC, IRD 224-CNRS 5290-UM, Institut de Recherche pour le Développement, 911 avenue Agropolis, 34394 Montpellier, Franceb Laboratoire d'entomologie médicale, Organisation de Coordination et de Coopération pour la Lutte Contre les Grandes Endémies en Afrique Centrale, Yaoundé, Cameroonc Université de Douala, Faculté de Médecine et des Sciences Pharmaceutiques, BP 2701, Douala, Cameroond UMI TransVIHMI 233, IRD 233-INSERM 1175-UM, Institut de Recherche pour le Développement, 911 avenue Agropolis, 34394 Montpellier, Francee W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USAf Emerging Pathogens Institute, Department of Infectious Diseases and Pathology, University of Florida, Gainesville, FL, USAg UMR 177 IRD-CIRAD INTERTRYP, TA A-17/G, Campus International de Baillarguet, 34398 Montpellier Cedex5, France

⁎ Corresponding author at: UMR MIVEGEC (IRD 224Recherche pour le Développement, 911 avenue Agropolicedex, France.

E-mail addresses: [email protected] (M.M. [email protected] (M.T. Tchioffo), nganobayi@[email protected] (P.H. Awono-Ambéné), [email protected] (C.B. Chesnais), [email protected]@ird.fr (T. de Meeûs), isabelle.morlais@ir

http://dx.doi.org/10.1016/j.meegid.2016.08.0251567-1348/© 2016 Published by Elsevier B.V.

a b s t r a c t
a r t i c l e i n f o
Article history:Received 23 May 2016Received in revised form 21 August 2016Accepted 22 August 2016Available online 23 August 2016

Progress in malaria control has led to a significant reduction of the malaria burden. Interventions that interrupttransmission are now needed to achieve the elimination goal. Transmission-blocking vaccines (TBV) that aimto prevent mosquito infections represent promising tools and several vaccine candidates targeting differentstages of the parasite's lifecycle are currently under development. Amosquito-midgut antigen, the anopheline al-anyl aminopeptidase (AnAPN1) is one of the lead TBV candidates; antibodies against AnAPN1 prevent ookineteinvasion. In this study, we explored the transmission dynamics of Plasmodium falciparum inmosquitoes fed withanti-AnAPN1monoclonal antibodies (mAbs) vs. untreated controls, and investigatedwhether the parasite genet-ic content affects or is affected by antibody treatment. Exposure to anti-AnAPN1 mAbs was efficient at blockingparasite transmission and the effect was dose-dependent. Genetic analysis revealed a significant sib-matingwithin P. falciparum infra-populations infecting one host, as measured by the strong correlation betweenWright's FIS and multiplicity of infection. Treatments also resulted in significant decrease in FIS as a by-productof drop in infra-population genetic diversity and concomitant increase of apparent panmictic genotyping propor-tions. Genetic differentiation analyses indicated that mosquitoes fed on a same donor randomly sampled blood-circulating gametocytes.We did not detect trace of selection, as the genetic differentiation between different do-nors did not decrease with increasing mAb concentration and was not significant between treatments for eachgametocyte donor. Thus, there is apparently no specific genotype associated with the loss of diversity undermAb treatment. Finally, the anti-AnAPN1 mAbs were effective at reducing mosquito infection and a vaccineaiming at eliciting anti-AnAPN1 mAbs has a strong potential to decrease the burden of malaria in transmis-sion-blocking interventions without any apparent selective pressure on the parasite population.

© 2016 Published by Elsevier B.V.

Keywords:Plasmodium falciparumAnopheles coluzziiAnti-AnAPN1 antibodiesGenetic structureMultiplicity of infectionSib-mating

1. Introduction

Plasmodium falciparum remains an important pathogen of humans,responsible for themajority of malaria cases and deaths in sub-Saharan

-CNRS 5290-UM), Institut des, BP64501, 34394 Montpellier

), [email protected] (L. Abate),.com (A.N. Bayibéki),@yahoo.fr (S.E. Nsango),du (R.R. Dinglasan),d.fr (I. Morlais).

Africa (WHO, 2015). The Malaria Control Strategy currently recom-mended by the WHO (WHO, 2015) relies on the use of artemisinin-based combination therapies (ACTs), intermittent preventive treatmentduring pregnancy (IPTp) and universal distribution of Long Lasting In-secticidal Nets (LLINs). However, widespread drug (Ariey et al., 2014;Dondorp et al., 2009) and insecticide (Briët et al., 2013; Ranson et al.,2009) resistances have hampered the efforts of malaria control pro-grams and new interventions are needed. In the current perspective ofmalaria elimination, transmission-blocking vaccines (TBVs) that targetthe parasite's sexual stages in the mosquitoes, represent promising ap-proaches (Nunes et al., 2014; Sauerwein and Bousema, 2015; Smith etal., 2011). Indeed, blocking Plasmodium development within the mos-quito would interrupt malaria transmission.

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139M.M. Sandeu et al. / Infection, Genetics and Evolution 45 (2016) 138–144

Several TBV candidates that target the mosquito stages of Plasmodi-um parasites are currently in pre- and clinical development (Sauerweinand Bousema, 2015). Themosquito-based TBV alanyl aminopeptidase N(AnAPN1)works in a similar fashion but targets amosquito receptor forthe parasite (Armistead et al., 2014; Atkinson et al., 2015). AnAPN1 is aglycoprotein expressed at the apical surface of themosquitomidgut ep-itheliumwith a role in ookinete invasion and is conserved across diver-gent anopheline species (Atkinson et al., 2015; Mathias et al., 2013).Antibodies against AnAPN1 were shown to block parasite transmissionof P. berghei, P. falciparum and P. vivax in different Old World species ofAnopheles mosquitoes, with parasite species-specific differences in theantibody efficacy (Armistead et al., 2014; Dinglasan et al., 2007;Mathias et al., 2013). The AnAPN1 structure was recently solved andused to map the relevant transmission-blocking epitopes and predictantibody functional activity (Atkinson et al., 2015). Although it hasbeen observed that complete blockade of P. falciparum developmentcan be achievedwith anti-AnAPN1 antibodies, the mechanism underly-ing the transmission-blocking activity remains elusive.

P. falciparum has a unique and complex cycle involving multiplecell divisions both in the mosquito and human hosts. Gametocytesare the sexual stages of the parasites that circulate in the peripheralblood of the vertebrate hosts and importantly, are the only parasiticstage that can infect mosquito vectors. Gametocytes are taken upwith the mosquito blood meal and transform into gametes. Fertiliza-tion between male and female gametes occurs in the midgut lumen,resulting in a diploid zygote. The zygote evolves into a motileookinete that traverses the midgut epithelium, reaches the basallamina, and develops into an oocyst that undergoes nuclear reduc-tion (meiosis). Intense multiplications within the oocyst lead to alarge number of haploid sporozoites that are released into the hemo-lymph when the mature oocyst ruptures. Sporozoites invade themosquito salivary glands and can be transmitted to a new vertebratehost during the next blood meal.

Oocysts represent a convenient stage for genetic studies: they can beindividually dissected from the midgut and they represent a diploidstage from which the parental genotypes can be inferred (Annan et al.,2007; Mzilahowa et al., 2007). In malaria endemic areas, human hostsharbor multiple genotypes of P. falciparum and recombination or“outcrossing” during sexual reproduction can occur between genetical-ly-distinct gametes in the mosquito (Annan et al., 2007; Morlais et al.,2015; Nkhoma et al., 2012; Paul et al., 1995). Several studies have re-ported high levels of inbreeding in oocyst populations from field-col-lected or laboratory-infected mosquitoes, indicating that sib mating(mating between related gametes) is common (Anderson et al., 2000;Annan et al., 2007; Morlais et al., 2015).

Host and parasite genetic heterogeneities in natural conditionsmight challenge the predicted efficacy of transmission-blocking in-terventions (TBIs). Efficacy of the RTS,S/AS01 vaccine was alreadyshown allele-specific and the protective effect depends on the para-site genotype at the target locus, the circumsporozoite protein(Neafsey et al., 2015). In malaria areas with high transmission,mixed-strain infections are common and competition between para-site strains influences the transmission of drug-resistant parasites inthe human host as well as the infection success in the mosquito vec-tor (Bushman et al., 2016; Morlais et al., 2015). Vaccines with incom-plete protection could also impose a selective pressure and favor thespread of escape/resistant genotypes (Gandon et al., 2001; Read etal., 2015).

In this study, we hypothesized that the administration of anti-APN1mAbs at suboptimal blocking concentrations would favor particular ge-notypes of P. falciparum. We tested this hypothesis in Cameroon, a ma-laria endemic area with high P. falciparum genetic diversity. Wegenotyped individual P. falciparum oocysts isolated from mosquitoeschallengedwith anti-AnAPN1mAbs and from control (untreated) mos-quitoes at seven microsatellite loci and compared the parasite geneticstructure in these two oocyst groups.

2. Materials and methods

2.1. Ethical statement

The asymptomatic gametocyte carriers were enrolled as volunteersafter their parents or legal guardians had provided informed consent.All procedures used in this study were approved by the Camerooniannational ethical committee (statements 2013/02/031/L/CNERSH/SP,2014/04/440/CE/CNERSH/SP and 2015/04/583/CE/CNERSH/SP).

2.2. Recruitment of P. falciparum gametocytes donors

Gametocyte carriers were identified among asymptomatic childrenaged between 5 and 11 years old who were attending primary schoolsin the Mfou district; a small town located 30 km South-East fromYaoundé, Cameroon. Blood samples were collected by finger prickfrom each volunteer and thick blood smears were stained with a 10%Giemsa solution and examined by light microscopy (×100 magnifica-tion) for the detection of asexual and sexual parasite stages. Gametocytedensity was estimated by counting parasite number against 1000 whiteblood cells (WBCs), assuming the standard number of 8000WBCs per μlof blood. Children with asexual parasitaemia exceeding 50 parasites/μlwere treatedwithDihydroartemisine –piperaquine (Malacur®) accord-ing to national guidelines.

2.3. Experimental infections of mosquitoes

We performed direct membrane-feeding assays (DMFAs) using fiveindependent, naturally-infected gametocyte donors. Our local mosquitocolony of An. coluzzii Ngousso was used for infection experiments. Foreach volunteer, gametocyte-infected blood was collected by venipunc-ture into heparinized vacutainer tubes. The blood was centrifuged at800 rcf at 37 °C for 5 min and the donor serum removed. Transmis-sion-blocking assays were performed using the 4H5B7 mAbs thatwere previously shown to block P. falciparum development in Anophelesmosquitoes (Atkinson et al., 2015). 4H5B7 mAbs were tested at finalconcentrations ranging from 0.5 to 50 μg/ml. Dilutions of 4H5B7 wereprepared in AB human serum from a non-immune donor and ABserum alone was used as control. For each assay, antibody preparationswere mixed at a 1:1 ratio with the packed red blood cell pellet (50% he-matocrit) from a single gametocyte-infected blood donor. Mixtureswere delivered into water-jacketed membrane feeders maintained at37 °C. Mosquitoes were allowed to feed for 30 min; unfed or partiallyfed females after this timewere removed from the study. Fully fedmos-quitoes were maintained in the insectary under standard conditions(27± 2 °C, 85± 5% RH, 12 h light/dark) and fed daily with a 6% sucroseuntil dissections.

2.4. Mosquito dissection and oocyst DNA extraction

At day seven post-infection, we used a batch of female mosquitoesfor each treatment (4H5B7-0.5, 4H5 B7-5, 4H5 B7-50 and AB) to evalu-ate the efficiency of the mAb concentrations at reducing parasite trans-mission. Mosquito midguts were dissected, stained with a 0.4%mercurochrome solution and examined under a Leica (Deerfield, IL)light microscope (×20) for oocyst counts. The infection prevalencewas computed as the percentage of mosquitoes with at least one oocystper midgut and the infection intensity as the arithmetic mean of oocystnumber in the feeding experiment.

The remaining mosquitoes were used for oocyst genotyping. Mid-guts were dissected at day 9 post-infection in a PBS/0.1% mercuro-chrome solution, transferred individually in 1.5 ml microtubescontaining 100 μl absolute ethyl alcohol and preserved at −20 °C(Annan et al., 2007). Oocysts were individually dissected from eachgut after gradual rehydration, consisting in 30 min in a 70% alcoholbath, 30 min in a 30% alcohol bath and then 30 min in distilled water.

140 M.M. Sandeu et al. / Infection, Genetics and Evolution 45 (2016) 138–144

Dissections were performed using a Leica DMIRB inverted microscope(Leica Microsystems). Each oocyst was carefully teased from the basallamina of the midgut epithelium with needles and transferred into anindividual 0.5 ml tube containing 10 μl of distilled water using aflame-narrowed Pasteur pipette. Particular carewas taken to avoid con-tamination during dissections; all dissecting tools were washed in 70%ethanol and replaced regularly. Parasite DNA from each oocyst was ex-tracted using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA) fol-lowing the manufacturer's instructions and stored at −20 °C untilgenotyping.

2.5. Gametocyte isolation and DNA extraction

For each blood donor, gametocyteswere isolated from100 μl venousblood filtrated through a LD column using the MACS system (MiltenyiBiotec) as previously described (Ribaut et al., 2008). DNAswere extract-ed using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA) followingthe manufacturer's instructions and stored at−20 °C until genotyping.

2.6. Microsatellite genotyping and genotype scoring

The genotyping was performed at 7 microsatellite loci (Table S1)and PCR amplifications were processed as previously described

Fig. 1. Schematic representation of the procedure for genetic analyses. F statistics were computeormosquito infra-population (MIF); oocysts inmosquitoes from a single blood donor, or donortreatment: T1, AB control; T2, 4H5B7 at 0.5 μg/μl; T3, 4H5B7 at 5 μg/μl. ID: gametocyte donor.

(Anderson et al., 2000; Annan et al., 2007). Fluorescence-labeled PCRproducts were sized on an ABI Prism 3100 genetic analyzer (AppliedBiosystems, Foster City, CA), using the GeneScan 500 LIZ dye as internalstandard. Alleles were assigned using the GeneMapper software (Ap-plied Biosystems, Foster City,CA). The same protocol was used to ampli-fy DNAs from gametocytes and oocysts. Gametocyte genotyping wasused to determine themultiplicity of infection (MOI), whichwas scoredas the maximum allele number at the most polymorphic locus. Oocystgenotyping was used to compare the genetic structure of P. falciparumgroups in antibody-treated and non-treated mosquitoes fed on thesame blood donor.

2.7. Statistical analysis

The data were analyzed using Stata 12.0 software (StataCorp, Col-lege Station, TX, 2005) and GraphPad Prism version 6.07 (GraphPadSoftware). First, we described the arithmetic mean of mosquito infec-tion according to the treatment concentration for each donor. Second,we assessed the effect of the treatment concentration (anti-AnAPN1 ef-ficacy) by performing a generalized linear mixedmodel using a Poissondistribution and a random effect on the donors. The mAb effect wasassessed with the β-coefficients (and their significance) and themodelscompared the efficacy of each concentration with the AB treatment

d at three hierarchical levels of “population” structure: Oocysts within amosquitomidgut,infra-population (DIF); all oocysts infectingmosquitoes in each treatment group (TG). T for


(control arm). Finally, themedian incidence rate ratio was computed toassess the proportion of the global variance due to the blood donors(random effect), and the significance was estimated using a likelihoodratio test.

2.8. Genetic analysis

Genetic analysis was performed on individual oocysts isolated frommosquitoes fed on blood from the five donors and treated at differentmAb concentrations. For this analysis, we considered three hierarchicallevels of “population” structure (Fig. 1): (i) oocysts within a mosquitomidgut in each treatment group, or mosquito infra-population (MIF);(ii) oocysts in mosquitoes from a single blood donor, or donor infra-population (DIF); and (iii) all oocysts infecting mosquitoes in eachtreatment group (TG). Within each treatment, the contribution of MIFand DIF to the genetic structure was assessed with HierFstat 0.04-10(Goudet, 2005) package for R (R-Development-core-team, 2015). Auser-friendly explanation on how using thus package is described in(de Meeûs and Goudet, 2007). Significance of MIF effect was tested by1000 randomisations of oocysts among mosquito individuals from thesame donor with the G-based test (Goudet et al., 1996). The effect ofDIF was tested through 1000 randomisations of individual mosquitoesacross donors. These tests were undertaken separately for each treat-ment. Linkage disequilibrium (LD) between the seven loci was investi-gated through the G-based randomization procedure per pair of locusoverall subsamples. This was implemented using Fstat 2.9.4. (Goudet,2003) with 10,000 random re-associations of genotypes betweenlocus pairs. Observed heterozygosity (Ho) and Nei's unbiased estimatorof genetic diversity (Hs) (Nei, 1978) were computed with Fstat version2.9.4. (Goudet, 2003), Wright's FIS is a measure of inbreeding of individ-uals relative to inbreeding of subpopulations. It is therefore also a mea-sure of the deviation of heterozygosity from Hardy-Weinbergexpectations in subsamples. It varies from −1 (all individuals are het-erozygous for the same two alleles within each subpopulation) to +1(all individuals are homozygous with at least two alleles in all subpop-ulations) and equals 0 when all subpopulations conform to genotypicproportions expected under panmixia.We used the unbiased estimatorf of Weir and Cockerham (Weir and Cockerham, 1984) to estimate FISand tested its significance deviation from 0 through 10,000 permuta-tions of alleles among individuals within sub-populations with Fstat2.9.4. (Goudet, 2003). Wright's FST measures the inbreeding of sub-

Fig. 2. Effect of anti-APN1 exposure on Plasmodium falciparum infection in the mosquito. The anAB serum; 0.5, 5 and 50, concentrations of 4H5B7in the bloodmeal in μg/μl. The highest concentarithmetic means of oocyst numbers, and error bars 95%CIs.

populations relative to inbreeding of the total population. It is thus ameasure of the degree of subdivision (genetic differentiation) acrosssub-populations. A value around 0 suggests an absence of subdivision(no genetic differentiation), while positive values reflects the effect ofsubdivision, with a maximum of 1 when all subsamples are fixed forone or the other allele. FST was estimated with Weir and Cockerham'sunbiased θ with Fstat and significance of differentiation between sub-populations was tested with the G based test (Goudet et al., 1996) and10,000 randomizations of individuals between subpopulations. 95%confidence intervals were computed by 5000 bootstrap simulationsover loci. We tested putative genetic changes between control (AB)and 4H5B7concentrations in each blood donor. We used Tériokhin'sgeneralized binomial procedure (Teriokhin et al., 2007) implementedinMultiTest v 1.2 software (deMeeûs et al., 2009) to combine indepen-dent tests over the five donors for each of the three paired comparisons.When b4 tests were combined, the whole series of P-values instead ofhalf of it was used, as recommended (deMeeûs, 2014). The significancelevels of non-independent series (linkage disequilibrium tests andpaired differentiation tests) were adjusted with the sequentialBonferroni correction (e.g. de Meeûs, 2014).

Normalised value of FST was estimated with Hedrick's method(Hedrick, 2005) as FSTʹ= FST / (1−Hs). Global decrease of FISwith treat-ment of each patient was tested with Page's trend test (Siegel andCastellan, 1988).

The relationship between FIS, the effect of donor, the multiplicity ofinfection (MOI) and the treatment intensity were tested with general-ized linear models with a Gaussian error. The complete models werethen simplified by a stepwise procedure. This was undertaken withRcmdr package (Fox, 2005, 2007) for R. The minimal model was thentested with Spearman's rank correlation tests with Rcmdr.

3. Results and discussion

The effect of anti-AnAPN1 mAb exposure was measured for five ga-metocyte donors. Mean of oocysts per mosquito varied from 5.4 to 11.4in non-exposed groups and oocyst distribution was heterogeneous,ranging from zero to 49 (Fig. 2 and Table 1). Anti-AnAPN1 mAb treatedmosquitoes were significantly less infected than untreated ones: mos-quito infection intensity decreased significantlywith increasing concen-trations of mAbs for the 5 experiments and 50 μg/μl was blocking for allthe 5 gametocyte donors (Fig. 2 and Table 1). This then confirmed our

tibody treatment is given for each gametocyte donor (ID code: C201 to C219): AB, controlration, 50 μg/μl, was blocking for all the 5 gametocyte donors. Horizontal lines indicate the

Table 3Hierarchical F statistics for each mAb treatment computed by HierFstat.

mAb Between donors Within donors

Treatment FST P-value FST-ID P-value

AB 0.604 0.001 −0.006 0.7154H5-0.5 0.645 0.001 −0.012 0.834H5-5 0.611 0.001 0.004 0.378

Table 1Mosquito infection parameters for each gametocyte donor and treatment.

Donor Treatment N Oocyst meana Range Prevalence (%)

C201 AB control 80 5.4 0–27 81.20.5 μg/μl 54 5.6 0–20 83.35 μg/μl 75 2.1 0–15 62.750 μg/μl 40 0 0–0 0


C207 AB control 65 8.4 0–29 89.20.5 μg/μl 56 5.2 0–24 67.85 μg/μl 35 4 0–16 8050 μg/μl 50 0 0–0 0



a Arithmetic mean of oocyst number per mosquito.


previous data on the transmission-blocking activity of the 4H5B7 mAbagainst field isolates of parasites from Cameroon (Atkinson et al.,2015). Infection intensities decreased by about one third at 0.5 μg/μl(β = −0.29; Table S2) and were roughly halved at 5 μg/μl(β = −1.17; Table S2). The blood donors explained 16% of the globalvariance of themAb efficacy on the mosquito infection intensity (likeli-hood ratio test for random effect, P-value b 0.001). Hence, the effect ofmAb treatment was not homogeneous between blood donors and thissuggests that the asexual parasites infecting the donor (genetic contentor density) affect differently themAb efficacy. Gametocyte donors wereidentified in an area of intense transmission of malaria where hostsmostly carry mixed-strain infections (Morlais et al., 2015; Nsango etal., 2012). We genotyped the gametocyte-only populations and indeedfound that all the parasite donors were infected by multiple genotypesof P. falciparum (Table 2).

A total of 1457 individual oocysts were processed for microsatellitegenotyping and 8% (116) failed to amplify. The 1341 remaining oocysts

Table 2F statistics per gametocyte donor and per mAb treatment.

Gametocytedonor

Gametocytedensity

MOI indonor

mAbtreatment

MOI Number ofguts

Number ofoocysts

H

C201 111 6 AB 7 18 95 04H5B7-0.5 5 8 52 04H5B7-5 4 17 67 0

C204 93 9 AB 4 16 197 0

4H5B7-0.5 3 20 151 04H5B7-5 2 11 36 0

C207 42 2 AB 4 15 96 0

4H5B7-0.5 3 14 101 04H5B7-5 1 13 70 0

C214 138 3 AB 5 13 97 04H5B7-0.5 3 12 91 04H5B7-5 3 13 84 0

C219 106 3 AB 2 9 86 0

4H5B7-0.5 5 16 79 0

4H5B7-5 2 8 39 0

Overall AB 4.6 71 571 04H5B7-0.5 3.6 70 474 04H5B7-5 2.4 62 296 0

used for population genetic analyses were isolated from 203 mosquitomidguts (Table S3); their distribution per midgut and gametocytedonor for eachmAb treatment is provided in Table 2. Linkage disequilib-rium was significant for all 21 pairs of loci, with P b 0.05 for each pairafter Bonferroni correction in the AB control groups. This indicatesstrong linkage between lociwithinmosquitoes from a samegametocytedonor, which constrains to increased prudence about interpreting am-biguous P-values based on multilocus statistics. There was no effect ofindividual mosquitoes in the genetic structure of P. falciparum oocysts(mean FST-ID ~ 0, P-value N0.378; Table 3), which is in agreement withprevious reports (Morlais et al., 2015). Each mosquito randomly sam-pled gametocytes from the blood donor and mosquitoes fed from thesame donor harboured a representative sample of the donor'sinfrapopulation, mosquito level was then ignored in the followinganalyses.

The effect of donor was very strong and significant (FST ≈ 0.6, P-value = 0.001 for each treatment; Table 3) and genetic differentiationdid not decreasewithmAb concentration, aswould be expected if a par-ticular genotypewas selected by the treatment, nor increased as expect-ed because subdivision was already extreme between control donors(FSTʹ ~ 0.8). Hence, even if the number of oocysts decreases with mAbconcentration, the remaining oocysts are still representative of whatthedonor initially harboured. In addition, the overall estimates of genet-ic differentiation between treatments for each parasite donor were notsignificant (Table S4) and no tendency for increased differentiationwith treatment dose appeared. This confirms that mosquitoes fed on asame gametocyte donor randomly sampled blood-circulating parasitesand treatments randomly blocked oocysts. Differences may howeverexist at the quantitative level and minority genotypes may be favouredin the mosquitoes. In the present work, the genotyping procedures didnot allow quantifying the different co-infecting parasite clones in the

o Hs FIS-ID (95% CI) P-value FST-ID (95% CI) P-value

.139 0.261 0.49 (0.252–0.608) 0.001 0.022 (−0.014–0.043) 0.435

.23 0.272 0.156 (0–0.3) 0.025 0.025 (−0.011–0.084) 0.085

.203 0.259 0.266 (0.198–0.476) 0.001 0.008 (−0.032–0.04) 0.218

.353 0.367 0.053(−0.048–0.208)

0.028 −0.001 (−0.021–0.004) 0.768

.298 0.355 0.141 (0.048–0.035) 0.001 −0.002 (−0.002–0.035) 0.475

.336 0.386 0.161(−0.004–0.215) 0.104 −0.041 (−0.09–0.015) 0.702

.011 0.015 0.29 (0.035–0.749) 0.008 −0.035(−0.064–(−0.025))

0.74

.006 0.007 0.026 (0.023–0.029) 0.999 −0.037 (−0.041–0.033) 0.937

.000 0.000 0.000 (n/a) n/a 0.000 (n/a) n/a

.288 0.465 0.373 (0.223–0.541) 0.001 −0.014 (−0.035–0.007) 0.708

.491 0.49 0.056 (−0.095–0.18) 0.032 −0.031(−0.05–(−0.013)) 0.805

.442 0.43 −0.012(−0.161–0.131)

0.745 0.015 (−0.006–0.03) 0.3

.223 0.24 0.102(−0.019–0.239)

0.025 0.009 (−0.022–0.039) 0.233

.166 0.192 0.071 (0.04–0.189) 0.013 −0.016(−0.025–(−0.009))

0.596

.318 0.315 0.008(−0.127–0.178)

0.52 0.005 (−0.012–0.022) 0.352

.198 0.267 0.216 (0.09–0.337) 0.001 −0.006 (−0.032–0.014) 0.577

.227 0.257 0.1 (0.005–0.189) 0.001 −0.013 (−0.026–0.013) 0.580

.249 0.267 0.096 (−0.02–0.238) 0.001 −0.003 (−0.028–0.022) 0.315

Fig. 3. Mean inbreeding levels within the donor infra-populations (FIS-ID) in the differenttreatment groups. The page test for ordered alternatives is significant (P-value = 0.05).


bulked gametocyte populations and this will have to be furtherinvestigated.

FIS-ID were then calculated for the three different treatment groups,AB, 4H5B7-0.5 and 4H5B7-5 (Table 2). The average FIS-ID were: 0.216(95% CI: 0.053–0.491, P b 10−3) in control group, 0.100 (95% CI:−0.007–0.156, P b 10−3) in 4H5B7-0.5 group and 0.096 (95% CI:−0.0012–0.266, P b 10−3) in 4H5B7-5 group (Fig. 3). FIS-ID decreasedas the antibody concentration increased, there is a negative correlationbetween FIS-ID and mAb treatment and the trend is just significant overall gametocyte donors (page's trend test, P-value = 0.05). Mean in-breeding levels relative to subpopulations (donor level) were loweredin mosquitoes exposed to anti-AnAPN1 mAbs, FIS-ID were no more sig-nificant at the highest non-blocking dose (5 μg/ml) for 4 out of 5blood donors (Table 2). Regression approaches revealed that MOI andtreatment were collinear as indeed MOI significantly decreased withmAb concentration (R2 = 0.29, P-value = 0.033). MOI then explainsmost of FIS-ID variation across donors and treatments (R2=0.58), the re-lationship is positive and highly significant (P-value = 0.013, Fig. 4).Thus, the more divergent gametes there is in an infra-population, thegreater deviation from Hardy-Weinberg will be. This translates into anobvious systematic sib-mating within P. falciparum infra-populations.Our results indicates that only closely related genotypes fuse to formoo-cysts in a diverse infra-population, while they mate randomly when amosquito is invaded by closely related gametes (lowMOI). And this ex-plains the apparent negative correlation between FIS-ID and mAb con-centration. Let us consider FIS = (QI − QS) / (1 − QS) (Cockerham,

Fig. 4. Correlation between multiplicity of infection and FIS-ID. Each dot represents afeeding experiment. Multiplicity of infection is positively correlated to the FIS-ID.

1969; Cockerham, 1973), where Q represents identity probabilities be-tween alleles in the same individual (I) or between individuals fromthe same sub-population (S). Since the highest treatment concentrationcan lower the gametocyte genetic diversity (MOI decreases) withoutchanging the within oocyst diversity (homozygosity does not change),then the treatment will increase QS without altering QI. Hence, FIS-ID isexpected to artificially decrease to some extentwith the treatment con-centration and does so in a just significantway thatmay be attributed toa by-product of sib-mating and maybe also of linkage across loci.

In conclusion, the anti-AnAPN1 mAbs efficiently lower the numberof oocysts that can be transmitted by mosquitoes. Treatment has alsoan effect on the diversity of genotypes that can be transmitted but in anonspecific way, as we did not detect selection on escaping genotypes.Interestingly, the significant positive correlation of FIS with MOI indi-cates that P. falciparum gametes preferentially mate between close rela-tives. Indeed, the strong and significant positive correlation between FISand MOI can only be explained either by preferential mating betweenrelated gametes or a greater success of highly related gametocyte pop-ulation while infecting mosquitoes, with similar consequences. Ourfinding need further investigations because it might come from the co-existence of separated lineages, genomically adapted to specific param-eters of their environment and not ready to break some specific genecombinations that allow their spread and maintenance. The anti-AnAPN1mAbs were effective at reducingmosquito infection and a vac-cine aiming at eliciting anti-AnAPN1 mAbs has a strong potential to de-crease the burden of malaria in transmission-blocking interventionswithout any apparent selective pressure on the parasite population.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.meegid.2016.08.025.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

IM and RRD conceived and designed the concept of the project.MMS, LA, MTT, ANB, SEN and IM collected samples. MMS, LA and MTTperformed the lab work. MMS, CBC and TDM analyzed the data. PHAA,SEN, RRD, TDM and IM contributed reagents/materials/analysis tools.MMS, TDM, and IM wrote the manuscript. All authors read and ap-proved the final manuscript.

Acknowledgments

This work was supported by funds from the Institut de Recherchepour le Développement (IRD) and the UE-ERC Malares (grant agree-ment 260918). We are grateful to volunteers from Mfou primaryschools and their parents or guardians for participating in this study,to the medical team from the Mfou Hospital for assistance in the fieldand to the technician staff from the IRD-OCEAC laboratory for P.falciparum infections and mosquito dissections. MMS was supportedby a fellowship from the IRD.

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Impact of exposure to mosquito transmission-blocking antibodies on Plasmodium falciparum population genetic structure1. Introduction2. Materials and methods2.1. Ethical statement2.2. Recruitment of P. falciparum gametocytes donors2.3. Experimental infections of mosquitoes2.4. Mosquito dissection and oocyst DNA extraction2.5. Gametocyte isolation and DNA extraction2.6. Microsatellite genotyping and genotype scoring2.7. Statistical analysis2.8. Genetic analysis

3. Results and discussionCompeting interestsAuthors' contributionsAcknowledgmentsReferences

infection, genetics and evolution · plasmodium falciparum anopheles coluzzii anti-anapn1...

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